The herbicide ketoclomazone inhibits 1-deoxy-D-xylulose 5-phosphate synthase in the 2-C-methyl-D-erythritol 4-phosphate pathway and shows antibacterial activity against Haemophilus influenzae

Abstract

Two distinct metabolic pathways have been elucidated for the formation of isopentenyl diphosphate and dimethylallyl diphosphate, essential metabolic precursors for isoprenoid biosynthesis: the mevalonate pathway, found ubiquitously in mammals, and the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, found in most bacteria. As the MEP pathway is absent from mammals, all MEP pathway enzymes represent effective targets for the development of antibacterial drugs. In this study, we found that a herbicide, ketoclomazone, exhibited antibacterial activity against a pathogenic bacterium, Haemophilus influenzae, with an MIC value of 12.5 lg ml À1 and that antibacterial activity was suppressed by adding 1-deoxyxylulose, a free alcohol of 1-deoxy-D-xylulose 5-phosphate (DXP). DXP is an MEP pathway intermediate synthesized from pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) by the action of DXP synthase. Thus, we investigated the enzyme kinetics of DXP synthase of H. influenzae (HiDXS) to elucidate an inhibitory mechanism of ketoclomazone on HiDXS. The dxs gene was cloned from H. influenzae and overexpressed in Escherichia coli, and the enzyme was purified to homogeneity. The purified HiDXS was a soluble dimeric 70-kDa protein. Steady-state kinetic constants for HiDXS were calculated, and Lineweaver-Burk plots were consistent with a ping-pong bi bi mechanism. The kinetics of inhibition by ketoclomazone suggested that ketoclomazone binds to an unidentified inhibitor-binding site that differs from both the pyruvate-binding site and the D-GAP-binding site on DXP synthase. These data reveal the inhibitory mechanism of ketoclomazone on DXP synthase.

1-Deoxy-d-xylulose 5-Phosphate Synthase Catalyzes a Novel Random Sequential Mechanism

“…The distinction between the previously proposed mechanisms (12,15) and the random sequential mechanism proposed here is difficult on the basis of kinetic analyses alone. The kinetic analysis presented here supports the notion that DXP synthase proceeds via a random sequential mechanism and highlights interesting differences with previously reported kinetic studies carried out on R. capsulatus DXP (12) and studies carried out on E. coli and H. influenzae DXP synthase (15).…”

“…A, ordered mechanism where pyruvate binds tightly and essentially irreversibly to DXP synthase (12). B, ping-pong mechanism where CO 2 liberation occurs prior to D-GAP binding (15).…”

“…Using immobilized enzyme and substrate, the authors determined substrate binding constants and suggested that the observed 1.7-fold enhancement in binding of D-GAP to DXP synthase in the presence of soluble pyruvate provides further support for an ordered mechanism. A conflicting report (15) proposes, on the basis of steady-state kinetics, that Escherichia coli and Haemophilus influenzae DXP synthase operate via a classical ping-pong mechanism in a manner similar to other ThDP-dependent enzymes (Fig. 2B).…”

Arabidopsis J-Protein J20 Delivers the First Enzyme of the Plastidial Isoprenoid Pathway to Protein Quality Control

“…Changes in DXS activity cause altered sensitivity to clomazone (CLM), a specific DXS inhibitor (Zeidler et al, 2000;Carretero-Paulet et al, 2006;Matsue et al, 2010). In Arabidopsis, germination and growth in the presence of CLM result in concentration-dependent bleaching and a concomitant inhibition of true leaf development ( Figure 3A), likely due to a reduced production of plastidial isoprenoid products, such as chlorophylls, carotenoids, and MEP-derived hormones (Figure 1).…”

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

“…In this study we describe the active site architecture of 1-deoxy-D-xylulose 5-phosphate synthase, a target for antimicrobial agents, herbicides, and antimalarial drugs [22] [23]. A role in catalysis has been assigned to conserved amino acid residues and a model of the catalytic site has been generated which can be of great interest to predict drug-enzyme interactions.…”